1. Field of the Invention
This invention refers to a polypeptide having inducing activity for the defence against biotic stress in plants, nucleotide sequence encoding the same, microorganism, compositions and methods. More specifically, this invention refers to a subtilisin polypeptide derived from Acremonium strictum, wherein such polypeptide has elicitory activity. According to a particular embodiment of the invention, the polypeptide is codified by nucleotide sequence SEQ ID NO: 1 or sequences being at least 90% homologous to the same, and has the amino acid sequence SEQ ID NO: 2 or sequences being at least 90% homologous to the same, wherein such polypeptide has elicitory activity.
2. Brief Description of the Related Art
Strawberry is a fruit with remarkable characteristics, which can be eaten fresh, dried, frozen or canned. From an economic point of view, strawberry is a very important culture because of the number of input and technology used, apart from the added value it can achieve due to the fact it can be industrialized and exported.
One of the main problems of producing strawberries is the great number of diseases and pests (biotic stress) that attack the cultivars thereof, which result in limitation to the fruit's production and quality. The main cause of biotic stress for strawberries is fungal diseases.
There are many diseases, the most frequent of which are as follows:
Root and crown rot: several diseases caused by fungi among the genus Phytophthora, Rhizoctonia and Colletotrichum (anthracnose).
Leaf diseases: Xanthomonas fragariae (angular leaf spot); Gnomonia comari (foliar spot); Mycosphaerella fragariae or Ramularia (leaf spot); Diplocarpon earliana (leaf burn or spot); Dendrophoma/Phomopsis obscurans (stain); Sphaerotheca macularis (oidium); Colletotrichum spp. (anthracnose).
Flower and fruit diseases: Rhizopus stolonifer (rot); Botrytis cinerea (grey mould); Colletotrichum spp. (anthracnose).
Strawberry anthracnose, caused by a Colletotrichum fungal complex, is definitely the disease that most adversely affects the culture thereof, attacking almost every organ in the plant and originating great loss both in fruit production and greenhouses (seedling production), mainly in tropical and subtropical agro-climatic regions.
As a result of the wide pathogenic diversity (at least three different species), almost all kinds of strawberries are sensitive to anthracnose. In terms of culture's genetic enhancement, it is difficult to combine in a single variety genes conferring certain characteristics regarding production, greenhouse management, and resistance to diseases, and even more difficult to a disease caused by different pathogens such as anthracnose.
Culture management together with chemical treatments help reducing anthracnose incidence. However, the excessive use of agrochemicals has a high impact on the ecosystem's degradation and on farm worker's health, as well as on water pollution and in the content of toxic residues in food, apart from favouring the origination of fungicidal-resistant fungi strains and having direct effects on the elimination of pests' natural enemies. The excess of fungicidal residues in the fruit can cause serious problems when marketing or exporting strawberries for not meeting the quality standards required by local authorities or importing markets.
There is currently a major global agreement as to promoting, in general, responsible or sustainable agriculture through the development of productive systems integrating low-environmental impact agronomic procedures (integrated production). Among the different biological approaches encompassed within the term biotechnology, the use of varieties with increased resistance (resulting from conventional or molecular biotechnology-assisted genetic enhancement) as well as Biological Control Agents (BCA) is included.
BCAs may (i) act directly on the pathogen such as in the case of “antagonist microorganisms”; (ii) exert an indirect action by interacting in the host plant, providing protection against a disease, either by: hypovirulence transmission to virulent races thereby neutralizing such disease, or (iii) by activating the plant's defense mechanisms, known as “Induced Resistance” or “preimmunization”.
Plants defend themselves from potential invading pathogens either through morphological structures acting as physical barriers that inhibit the pathogen's entrance and development, or by biochemical reactions occurring in different plant tissues producing toxic substances for the pathogen, or creating conditions that inhibit the microorganism's entrance and development in the host. Such defenses may be either part of the plant being a non-specific protection against a wide range of microorganisms or induced in the presence of the attacking pathogen. In this latter case, plants as well as other living beings activate their own defense systems as they recognize a pathogenic-microorganism-derived molecule or else as they detect any molecule generated during the pathogen's invasion, any of them being referred to as defense “inducer” or “elicitor”.
In plant/pathogen interactions, inducing molecules alert of the presence of the invading pathogen when the pathogen is recognized by the host plant (Nürnberger, 1999. Cell Mol. Life Sci. 55, 167-182). In early recognition of the elicitors of the attacking pathogen, the plant produces a rapid activation of its defence mechanisms, which block the infection, stopping the pathogen's progress. In this case, the plant/pathogen interaction is said to be incompatible because it does not result in a disease, the phytopathogen strain being defined as non-virulent (Keen, 1990. Annu. Rev. Genet. 24, 447-463). In contrast, the plant develops the disesease when it is unable to detect the pathogen's aggression or when it detects it late (the elicitors thereof), and in spite of triggering certain defence mechanisms, they are not sufficient to timely stop the invasion, thus the plant/pathogen interaction is said to be compatible and the strain is deemed virulent.
During a fungal infection, plants can recognize the aggressor via a set of elicitors. Some of the inducing molecules are derived from the pathogen (non-self factors) and can be present at the fungal surface (e.g., chitin and glucan fragments) or secreted by the pathogen (e.g., avirulence proteins); while other molecules are generated by the plant during the fungal invasion (known as self factors), such as in the case of plant cell wall fragments (e.g., oligogalacturonates, chitin, heptaglucans, monosilated glycopeptides) released from polymeric precursors by action of the invading pathogen's hydrolytic enzymes (Knogge, 1996. The Plant Cell 8, 1711-1722). Thus, elicitors can be classified as pre-formed compounds, which are present on the pathogen's surface, or as induced, as when synthesized during the interaction between the pathogen and the host plant.
In general, the pathogen recognition and the subsequent activation of resistance responses to disease in plants can occur at a species level (e.g., species or non-host resistance, or cultivar non-specific host resistance, or innate immunity) or at a genotype level (cultivar specific host resistance). A cultivar's specific resistance is expressed in a given cultivar against one or a reduced number of pathogenic races and represents what is known as “gene to gene” response, being genetically determined by the complementary pair codified by the pathogen avirulence (Avr) gene and the product from a plant resistance (R) gene.
Thus, when the AVR protein is directly or indirectly recognized by a resistant host plant, it acts as a defense “specific elicitor”, and can be detected by the plant surveillance system. However, innate immunity is the prevailing resistance form in all plants species. In this response, a great variety of products associated to so-called “general elicitor” microorganisms, induce the defense response in many plant species and do not depend on a specific cultivar. The term “Pathogen-Associated Molecular Pattern” (PAMP) refers to any molecule capable of activating the plant defense system, and it can be found in a wide range of pathogens (Bent and Mackey, 2007. Annu. Rev. Phytopathol. 45, 399-436). Kamoun (Kamoun, 2006. Annu. Rev. Phytopathol. 44, 41-60) reports the elicitors produced by plant pathogenic oomycetes fungi, while Stergiopoulos et al. (Stergiopoulos and de Wit, 2009. Annu. Rev. Phytopathol. 47, 233-263) describe fungal avirulence proteins reported so far.
After the recognition of the elicitor, a series of cytological shifts and biochemical responses in plant cells have been identified. In biochemical terms, it can be said that an inducer interacts with one receptor on the cell surface which detects the extracellular signal converting it in intracellular signals, the transduction of which imply (a) ionic trans-membrane fluids (i.e. entrance of Ca2+, H+ and Cl−,); (b) production of reactive oxygen species (ROS) such as H2O2, O2−, etc., toxic for the cells, producing “oxidative burst”; (c) nitric oxide production (NO); c) phosphorylation/dephosphorylation of mitogen-activated protein kinases (MAPKs) and other calcium dependant protein kinases (CDPKs).
These signals give rise to early defense responses at the infection site, and late defence responses in distant plant areas.
Local defense responses to elicitors imply regulating several genes, which contribute to generate protective physiological conditions against invading pathogens. At the infection site, such responses include generation of reactive oxygen species (ROS), rapid accumulation of several enzymes and metabolites, such as for example, proteins involved in the production of signals, such as salicylic acid (SA), jasmonates and/or ethylene, and of enzymes related to phenylpropanoid metabolism (PAL: phenyl ammonium-lyase; CHS: chalcone synthase; etc.) and phytoalexin biosynthesis, low molecular weight secondary metabolites having antimicrobial activity, and the so-called PR proteins (Pathogenesis Related Proteins). Such defense is restricted to the area surrounding the pathogen penetration site.
In some cases, cells at the infection site can undergo a process of cell death, which usually becomes visible as a hypersensitive response. Hypersensitive Response (HR) consists in the rapid and localized death of the host cells invaded by the pathogen, by a necrosis phenomenon or programmed cell death (PCD). This phenomenon is associated with reinforcement in the cell wall of affected cells by local lignifications and callose accumulation, cross-linking of hydroxyproline-rich glycoproteins (HRGP), activation of the enzymes implied in molecule cross-linking as a plant strategy to limit the colonization to infection sites.
Salicylic acid (SA), is a plant hormone which among other functions inhibits catalase aggravating oxidative stress, and also coordinates the expression of PR protein subgroup which are divided in three classes: chitinases, glucanases and chitin binding proteins. In summary, to kill or successfully stop an invading microorganism a special and accurately timely coordination of induced defense responses is required.
A local infection often leads to induction of similar defense responses in non-infected plant tissues thus resulting in resistance to subsequent infections (Kuć, 1982. BioScience 32, 854-860). This line of defense leads to the accumulation of proteins and hydrolytic enzymes throughout the organisms, thus being referred to as “systemic” (Hunt and Ryals, 1996. Crit. Rev. Plant Sci. 15, 583-606). It usually provides resistance to the primary inducing agent (virulent pathovar) and also to a wide range of other fungal, bacterial and viral pathogens (immunization). When this defense response is mediated by action of an avirulent pathogen, it is referred to as “Systemic Acquired Resistance” (SAR). Furthermore, systemic resistance may also be triggered by a rhizosphere non-pathogenic microorganism, and in this case it is referred to as “Induced Systemic Resistance” (ISR); or it may be induced by injuries (mechanical damage).
The different systemic defense responses associated with pathogen infections include induction of several PR genes, accumulation of phytoalexins, induction of EROs and micro-HR.
The foregoing suggests that the induction of a broad-spectrum systemic defense response (either SAR or ISR) can be used as “immunization” strategy in order to prevent or reduce crop diseases (Induced Resistance or IR). For strawberries, the use of IR for controlling Phytophthora spp. (Eikemo et al., 2003. Plant Dis. 87, 345-350) and Botrytis (Adikaram et al., 2002. Australasian Plant Pathology 31(3), 223-229) has been suggested. This host plant protection phenomenon which was originally referred to as “Cross-protection” was used as means of management of virus-caused diseases by prior inoculation with weak strains of the same virus, thus making that the virus inoculated first prevent the development of the subsequent virus. Then, it was apparent that it was also possible to increase resistance to fungal pathogen severe races by pre-inoculation with an avirulent genotype of the same fungus species or by application of a rhizosphere non-pathogenic microorganism; as mentioned above, this last phenomenon was called ISR as opposed to SAR.
Unlike the response induced by a rhizosphere non-pathogenic microorganism, field implementation of a SAR-like system, which implies direct plant infection with a live pathogenic microorganism, is not possible, since it poses serious problems, among which it is worth mentioning that it is possible that an avirulent strain used to protect a cultivar can produce the disease in other susceptible genotypes (Fulton, 1986. Annu. Rev. Phytopathol. 24, 67-81). A biotechnological option to solve this problem and induce resistance involves inactivating the pathogen and applying portions from different pathogenic cultures (non-pathogenic extracts) that retain inducing defense activity, which at the same time lose pathogenic potential, that is, using fractions of the inactivated pathogen containing the defense inducing agent(s). Having greater knowledge of the system will allow for the direct application of elicitor molecules derived from the plant/avirulent pathogen interaction, capable of providing resistance against diseases through the induction of a broad-spectrum systemic defense response (SAR elicitor), to the expression of such pathogen avirulent gene in transgenic plants.
Very few elicitors that can effectively induce resistance are currently known. In strawberries, registered biopesticides with elicitory action include harpin proteins (trade name: MESSENGER®), an alternative to the use of methyl bromide, which is effective against bacterial leaf spot, tristeza caused by bacteria, bacterial blight, certain fungal diseases; and chitosan (trade name: ELEXA-4®) which is active against woolly aphids and powdery “mildew” and grey mould. However, until now, no type of biological control method to control anthracnose (Colletotrichum spp.) in strawberries has been suggested.
In light of the above mentioned, having determined the lack of solutions with respect to the provision of new disease treatment and/or prevention methods, the inventors herein have identified a new protein excreted to the medium by Acremonium strictum, and having purified it to homogeneity. This protein acts as avirulence factor (elicitor), triggering different defense mechanisms immunizing the plant, thus making it resistant to diseases, such as anthracnose produced by Colletotrichum spp.